The invention relates to nucleic acid detection. More specifically, the invention relates to the determination of the presence or absence of multiple targeted, predetermined nucleic acid sequences in nucleic acid target/probe hybrids, and the various applications of their detection.
Methods to detect nucleic acids and to detect specific nucleic acids provide a foundation upon which the large and rapidly growing field of molecular biology is built. There is constant need for alternative methods and products. The reasons for selecting one method over another are varied, and include a desire to avoid radioactive materials, the lack of a license to use a technique, the cost or availability of reagents or equipment, the desire to minimize the time spent or the number of steps, the accuracy or sensitivity for a certain application, the ease of analysis, the need to detect multiple nucleic acids in one sample, or the ability to automate the process.
The detection of nucleic acids or specific nucleic acids is often a portion of a process rather than an end in itself. There are many applications of the detection of nucleic acids in the art, and new applications are always being developed. The ability to detect and quantify nucleic acids is useful in detecting microorganisms, viruses and biological molecules, and thus affects many fields, including human and veterinary medicine, food processing and environmental testing. Additionally, the detection and/or quantification of specific biomolecules from biological samples (e.g. tissue, sputum, urine, blood, semen, saliva) has applications in forensic science, such as the identification and exclusion of criminal suspects and paternity testing as well as medical diagnostics.
Some general methods to detect nucleic acids are not dependent upon a priori knowledge of the nucleic acid sequence. A nucleic acid detection method that is not sequence specific, but is RNA specific is described in U.S. Pat. No. 4,735,897, where RNA is depolymerized using a polynucleotide phosphorylase (PNP) in the presence of phosphate or using a ribonuclease. PNP stops depolymerizing at or near a double-stranded RNA segment. Sometimes double-stranded RNA can occur as a type of secondary structure RNA, as is common in ribosomal RNA, transfer RNA, viral RNA, and the message portion of mRNA. PNP depolymerization of the polyadenylated tail of mRNA in the presence of inorganic phosphate forms ADP. Alternatively, depolymerization using a ribonuclease forms AMP. The formed AMP is converted to ADP with myokinase, and ADP is converted into ATP by pyruvate kinase or creatine phosphokinase. Either the ATP or the byproduct from the organophosphate co-reactant (pyruvate or creatine) is detected as an indirect method of detecting mRNA.
In U.S. Pat. No. 4,735,897, ATP is detected by a luciferase detection system. In the presence of ATP and oxygen, luciferase catalyzes the oxidation of luciferin, producing light that can then be quantified using a luminometer. Additional products of the reaction are AMP, pyrophosphate and oxyluciferin.
Duplex DNA can be detected using intercalating dyes such as ethidium bromide. Such dyes are also used to detect hybrid formation.
Hybridization methods to detect nucleic acids are dependent upon knowledge of the nucleic acid sequence. Many known nucleic acid detection techniques depend upon specific nucleic acid hybridization in which an oligonucleotide probe is hybridized or annealed to nucleic acid in the sample or on a blot, and the hybridized probes are detected.
A traditional type of process for the detection of hybridized nucleic acid uses labeled nucleic acid probes to hybridize to a nucleic acid sample. For example, in a Southern blot technique, a nucleic acid sample is separated in an agarose gel based on size and affixed to a membrane, denatured, and exposed to the labeled nucleic acid probe under hybridizing conditions. If the labeled nucleic acid probe forms a hybrid with the nucleic acid on the blot, the label is bound to the membrane. Probes used in Southern blots have been labeled with radioactivity, fluorescent dyes, digoxygenin, horseradish peroxidase, alkaline phosphatase and acridinium esters.
Another type of process for the detection of hybridized nucleic acid takes advantage of the polymerase chain reaction (PCR). The PCR process is well known in the art (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). To briefly summarize PCR, nucleic acid primers, complementary to opposite strands of a nucleic acid amplification target sequence, are permitted to anneal to the denatured sample. A DNA polymerase (typically heat stable) extends the DNA duplex from the hybridized primer. The process is repeated to amplify the nucleic acid target. If the nucleic acid primers do not hybridize to the sample, then there is no corresponding amplified PCR product. In this case, the PCR primer acts as a hybridization probe. PCR-based methods are of limited use for the detection of nucleic acid of unknown sequence.
In a PCR method, the amplified nucleic acid product may be detected in a number of ways, e.g. incorporation of a labeled nucleotide into the amplified strand by using labeled primers. Primers used in PCR have been labeled with radioactivity, fluorescent dyes, digoxygenin, horseradish peroxidase, alkaline phosphatase, acridinium esters, biotin and jack bean urease. PCR products made with unlabeled primers may be detected in other ways, such as electrophoretic gel separation followed by dye-based visualization.
Multiplex PCR assays are well known in the art. For example, U.S. Pat. No. 5,582,989 discloses the simultaneous detection of multiple known DNA sequence deletions. The technique disclosed therein uses a first set of probes to hybridize to the targets. Those probes are extended if the targets are present. The extension products are amplified using PCR.
Fluorescence techniques are also known for the detection of nucleic acid hybrids, U.S. Pat. No. 5,691,146 describes the use of fluorescent hybridization probes that are fluorescence-quenched unless they are hybridized to the target nucleic acid sequence. U.S. Pat. No. 5,723,591 describes fluorescent hybridization probes that are fluorescence-quenched until hybridized to the target nucleic acid sequence, or until the probe is digested. Such techniques provide information about hybridization, and are of varying degrees of usefulness for the determination of single base variances in sequences. Some fluorescence techniques involve digestion of a nucleic acid hybrid in a 5xe2x80x2xe2x86x923xe2x80x2 direction to release a fluorescent signal from proximity to a fluorescence quencher, for example, TaqMan (Perkin Elmer; U.S. Pat. No. 5,691,146 and No. 5,876,930).
Enzymes having template-specific polymerase activity for which some 3xe2x80x2xe2x86x925xe2x80x2 depolymerization activity has been reported include E. coli DNA Polymerase (Deutscher and Kornberg, J. Biol. Chem., 244(11):3019-28 (1969)), T7 DNA Polymerase (Wong et al., Biochemistry 30:526-37 (1991); Tabor and Richardson, J. Biol. Chem. 265: 8322-28 (1990)), E. coli RNA polymerase (Rozovskaya et al., Biochem. J. 224:645-50 (1994)), AMV and RLV reverse transcriptases (Srivastava and Modak, J. Biol. Chem. 255: 2000-4 (1980)), and HIV reverse transcriptase (Zinnen et al., J. Biol. Chem. 269:24195-202 (1994)). A template-dependent polymerase for which 3xe2x80x2 to 5xe2x80x2 exonuclease activity has been reported on a mismatched end of a DNA hybrid is phage 29 DNA polymerase (de Vega, M. et al. EMBO J., 15:1182-1192, 1996).
A variety of methodologies currently exist for the detection of single nucleotide polymorphisms (SNPs) that are present in genomic DNA. SNPs are DNA point mutations or insertions/deletions that are present at measurable frequencies in the population. SNPs are the most common variations in the genome. SNPs occur at defined positions within genomes and can be used for gene mapping, defining population structure, and performing functional studies. SNPs are useful as markers because many known genetic diseases are caused by point mutations and insertions/deletions. Some SNPs are useful as markers of other disease genes because they are known to cosegregate.
In rare cases where an SNP alters a fortuitous restriction enzyme recognition sequence, differential sensitivity of the amplified DNA to cleavage can be used for SNP detection. This technique requires that an appropriate restriction enzyme site be present or introduced in the appropriate sequence context for differential recognition by the restriction endonuclease. After amplification, the products are cleaved by the appropriate restriction endonuclease and products are analyzed by gel electrophoresis and subsequent staining. The throughput of analysis by this technique is limited because samples require processing, gel analysis, and significant interpretation of data before SNPs can be accurately determined.
Single strand conformational polymorphism (SSCP) is a second technique that can detect SNPs present in an amplified DNA segment (Hayashi, K. Genetic Analysis: Techniques and Applications 9:73-79, 1992). In this method, the double stranded amplified product is denatured and then both strands are allowed to reanneal during electrophoresis in non-denaturing polyacrylamide gels. The separated strands assume a specific folded conformation based on intramolecular base pairing. The electrophoretic properties of each strand are dependent on the folded conformation. The presence of single nucleotide changes in the sequence can cause a detectable change in the conformation and electrophoretic migration of an amplified sample relative to wild type samples, allowing SNPs to be identified. In addition to the limited throughput possible by gel-based techniques, the design and interpretation of SSCP based experiments can be difficult. Multiplex analysis of several samples in the same SSCP reaction is extremely challenging. The sensitivity required in mutation detection and analysis has led most investigators to use radioactively labeled PCR products for this technique.
In the amplification refractory mutation system (ARMS, also known as allele specific PCR or ASPCR), two amplification reactions are used to determine if a SNP is present in a DNA sample (Newton et al. Nucl Acids Res 17:2503, 1989; Wu et al. PNAS 86:2757, 1989). Both amplification reactions contain a common primer for the target of interest. The first reaction contains a second primer specific for the wild type product which will give rise to a PCR product if the wild type gene is present in the sample. The second PCR reaction contains a primer that has a single nucleotide change at or near the 3xe2x80x2 end that represents the base change that is present in the mutated form of the DNA. The second primer, in conjunction with the common primer, will only function in PCR if genomic DNA that contains the mutated form of genomic DNA is present. This technique requires duplicate amplification reactions to be performed and analyzed by gel electrophoresis to ascertain if a mutated form of a gene is present. In addition, the data must be manually interpreted.
Single base extension (GBA(copyright)) is a technique that allows the detection of SNPs by hybridizing a single strand DNA probe to a captured DNA target (Nikiforov, T. et al. Nucl Acids Res 22:4167-4175). Once hybridized, the single strand probe is extended by a single base with labeled dideoxynucleotides. The labeled, extended products are then detected using calorimetric or fluorescent methodologies.
A variety of technologies related to real-time (or kinetic) PCR have been adapted to perform SNP detection. Many of these systems are platform based, and require specialized equipment, complicated primer design, and expensive supporting materials for SNP detection. In contrast, the process of this invention has been designed as a modular technology that can use a variety of instruments that are suited to the throughput needs of the end-user. In addition, the coupling of luciferase detection sensitivity with standard oligonucleotide chemistry and well-established enzymology provides a flexible and open system architecture. Alternative analytical detection methods, such as mass spectroscopy, HPLC, and fluorescence detection methods can also be used in the process of this invention, providing additional assay flexibility.
SNP detection using real-time amplification relies on the ability to detect amplified segments of nucleic acid as they are during the amplification reaction. Three basic real-time SNP detection methodologies exist: (i) increased fluorescence of double strand DNA specific dye binding, (ii) decreased quenching of fluorescence during amplification, and (iii) increased fluorescence energy transfer during amplification (Wittwer, C. et al. Biotechniques 22:130-138, 1997). All of these techniques are non-gel based and each strategy will be briefly discussed.
A variety of dyes are known to exhibit increased fluorescence in response to binding double stranded DNA. This property is utilized in conjunction with the amplification refractory mutation system described above to detect the presence of SNP. Production of wild type or mutation containing PCR products are continuously monitored by the increased fluorescence of dyes such as ethidium bromide or Syber Green as they bind to the accumulating PCR product. Note that dye binding is not selective for the sequence of the PCR product, and high non-specific background can give rise to false signals with this technique.
A second detection technology for real-time PCR, known generally as exonuclease primers (TaqMan(copyright) probes), utilizes the 5xe2x80x2 exonuclease activity of thermostable polymerases such as Taq to cleave dual-labeled probes present in the amplification reaction (Wittwer, C. et al. Biotechniques 22:130-138, 1997; Holland, P et al PNAS 88:7276-7280, 1991). While complementary to the PCR product, the probes used in this assay are distinct from the PCR primer and are dually-labeled with both a molecule capable of fluorescence and a molecule capable of quenching fluorescence. When the probes are intact, intramolecular quenching of the fluorescent signal within the DNA probe leads to little signal. When the fluorescent molecule is liberated by the exonuclease activity of Taq during amplification, the quenching is greatly reduced leading to increased fluorescent signal.
An additional form of real-time PCR also capitalizes on the intramolecular quenching of a fluorescent molecule by use of a tethered quenching moiety. The molecular beacon technology utilizes hairpin-shaped molecules with an internally-quenched fluorophore whose fluorescence is restored by binding to a DNA target of interest (Kramer, R. et al. Nat. Biotechnol. 14:303-308, 1996). Increased binding of the molecular beacon probe to the accumulating PCR product can be used to specifically detect SNPs present in genomic DNA.
A final, general fluorescent detection strategy used for detection of SNP in real time utilizes synthetic DNA segments known as hybridization probes in conjunction with a process known as fluorescence resonance energy transfer (FRET) (Wittwer, C. et al. Biotechniques 22:130-138, 1997; Bernard, P. et al. Am. J. Pathol. 153:1055-1061, 1998). This technique relies on the independent binding of labeled DNA probes on the target sequence. The close approximation of the two probes on the target sequence increases resonance energy transfer from one probe to the other, leading to a unique fluorescence signal. Mismatches caused by SNPs that disrupt the binding of either of the probes can be used to detect mutant sequences present in a DNA sample.
There is a need for alternative methods for the detection of a plurality of nucleic acid hybrids in a single sample. There is a demand for such methods that are highly sensitive. For example methods to determine viral load of multiple viruses in a single sample that are able to reliably detect as few as 10 copies of a virus present in a body, tissue, fluid, or other biological sample would be in high demand. There is a great demand for methods to determine the presence or absence of nucleic acid sequences that differ slightly from sequences that might otherwise be present. There is a great demand for methods to determine the presence or absence of sequences unique to a particular species in a sample. There is also a great demand for methods that are more highly sensitive than the known methods, quantitative, highly reproducible and automatable.
It would be beneficial if another method were available for detecting the presence of a sought-after, predetermined target nucleotide sequence or allelic or polynucleotide variant. It would also be beneficial if such a method were operable using a sample size of the microgram to picogram scale. It would further be beneficial if the various methods listed above were capable of providing multiple analyses in a single assay (multiplex assays). The disclosure that follows provides one such method.
A method of this invention is used to determine the presence or absence of a plurality of predetermined (known) nucleic acid target sequences in a nucleic acid sample. Such a method utilizes an enzyme that can depolymerize the 3xe2x80x2-terminus of an oligonucleotide probe hybridized to a nucleic acid target sequence to release one or more identifier nucleotides whose presence or absence can then be determined.
One embodiment of the invention contemplates a method for determining the presence or absence of a plurality of predetermined nucleic acid target sequences in a nucleic acid sample. Thus, the presence or absence of at least two predetermined nucleic acid target sequence is sought to be determined. This embodiment comprises the following steps.
A treated sample is provided that may contain a plurality of predetermined nucleic acid target sequences hybridized with their respective nucleic acid probes that include an identifier nucleotide in the 3xe2x80x2-terminal region. The treated sample is admixed with a depolymerizing amount of an enzyme whose activity is to release one or more identifier nucleotides from the 3xe2x80x2-terminus of a hybridized nucleic acid probe to form a treated reaction mixture. The treated reaction mixture is maintained under depolymerizing conditions for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release identifier nucleotides therefrom.
An analytical output is obtained by analyzing for the presence or absence of released identifier nucleotides, preferably such that the probe from which the nucleotide was released is distinguishable. The analytical output indicates the presence or absence of the nucleotide at the predetermined regions of the nucleic acid targets, and, thereby, the presence or absence of the nucleic acid targets.
It is contemplated that an analytical output of the methods of the invention can be obtained in a variety of ways. The analytical output can be ascertained by luminescence. In some preferred embodiments, analysis for released 3xe2x80x2-terminal region identifier nucleotides comprises the detection of ATP, either by a luciferase detection system (luminescence) or an NADH detection system (absorbance spectroscopy). In particularly preferred embodiments, ATP molecules are formed from the nucleotide triphosphates released by the depolymerizing step by a phosphate transferring step, for example using an enzyme such as NDPK (Nucleotide Diphosphate Kinase) in the presence of ADP. In some embodiments the ATP is amplified to form a plurality of ATP molecules. In the ATP detection embodiments, typically the enzyme (NDPK) for converting nucleotides and added ADP into ATP is present in the depolymerization reaction, and thus they are denoted as a xe2x80x9cone potxe2x80x9d method.
In an alternative embodiment, the analytical output is obtained by fluorescence spectroscopy. Use of a wide variety of fluorescence detection methods is contemplated. In one exemplary contemplated method, an identifier nucleotide includes a fluorescent label. In a multiplex analysis where it is desirable to distinguish which nucleic acid target sequences are present and which are absent, multiple types of labels can be used. An identifier nucleotide can be fluorescently labeled prior to, or after, release of the identifier nucleotide. It is also contemplated that other than a released identifier nucleotide contains a fluorescent tag. In such an embodiment, the release of nucleotides in a process of the invention is ascertained by a determination of a difference in the length of the polynucleotide probe, for example by capillary electrophoresis imaged by a fluorescent tag at the 5xe2x80x2 terminus of the probe or in a region other than the 3xe2x80x2 terminal region.
In an alternative embodiment, the analytical output is obtained by mass spectrometry. It is preferred here that an identifier nucleotide be a nucleotide analog or a labeled nucleotide and have a molecular mass that is different from the mass of a usual form of that nucleotide, although a difference in mass is not required. It is also noted that with a fluorescently labeled identifier nucleotide, the analytical output can also be obtained by mass spectrometry. It is also contemplated that the analysis of released nucleotides be conducted by ascertaining the difference in mass of the probe after a depolymerization step of a process of the invention.
In another alternative embodiment, the analytical output is obtained by absorbance spectroscopy. Such analysis monitors the absorbance of light in the ultraviolet and visible regions of the spectrum to determine the presence of absorbing species. In one aspect of such a process, released nucleotides are separated from hybridized nucleic acid and other polynucleotides by chromatography (e.g. HPLC or GC) or electrophoresis (e.g. PAGE or capillary electrophoresis). Either the released identifier nucleotides or the remainder of the probe can be analyzed for to ascertain the release of the identifier nucleotide in a process of the invention. In another aspect of such a process a label may be incorporated in the analyzed nucleic acid.
In another contemplated embodiment, a sample to be assayed is admixed with two or more nucleic acid probes under hybridizing conditions to form a hybridization composition. The 3xe2x80x2-terminal region of a nucleic acid probe hybridizes with partial or total complementarity to the nucleic acid target sequence when that sequence is present in the sample. The 3xe2x80x2-terminal region of the nucleic acid probe includes an identifier nucleotide.
The hybridization composition is maintained under hybridizing conditions for a time period sufficient to form a treated sample that may contain said predetermined nucleic acid target sequence hybridized with a nucleic acid probe. The treated sample is admixed with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3xe2x80x2-terminus of a hybridized nucleic acid probe to form a treated reaction mixture. The treated reaction mixture is maintained under depolymerizing conditions for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release identifier nucleotides therefrom.
The presence of released identifier nucleotides is analyzed to obtain an analytical output, the analytical output indicating the presence or absence of the nucleic acid target sequence. The analytical output may be obtained by various techniques as discussed above.
One method of the invention contemplates interrogating the presence or absence of a specific bases in their nucleic acid target sequences in a sample to be assayed, and comprises the following steps. Here, a hybridization composition is formed by admixing a sample to be assayed with a plurality of nucleic acid probes under hybridizing conditions. The sample to be assayed may contain a nucleic acid target sequence to be interrogated. The nucleic acid target comprises at least one base whose presence or absence is to be identified. The hybridization composition includes a plurality of nucleic acid probes that are each substantially complementary to a nucleic acid target sequence of interest and each probe comprises at least one predetermined nucleotide at an interrogation position, and an identifier nucleotide in the 3xe2x80x2-terminal region.
A treated sample is formed by maintaining the hybridization composition under hybridizing conditions for a time period sufficient for base pairing to occur for all probes when a probe nucleotide at an interrogation position is aligned with a base to be identified in its target sequence. A treated reaction mixture is formed by admixing the treated sample with an enzyme whose activity is to release one or more identifier nucleotides from the 3xe2x80x2-terminus of a hybridized nucleic acid probe to depolymerize the hybrid. The enzymes that can be used in this reaction are further discussed herein. The treated reaction mixture is maintained under depolymerizing conditions for a time period sufficient to permit the enzyme to depolymerize the hybridized nucleic acid and release an identifier nucleotide.
An analytical output is obtained by analyzing for the presence or absence of released identifier nucleotides. The analytical output indicates the presence or absence of the specific base or bases to be identified. The analytical output is obtained by various techniques, as discussed herein. Preferably, an identifier nucleotide is at the interrogation position. In one preferred embodiment, one is able to determine if at least one of the plurality of targets is present in the sample. In an alternative preferred embodiment, one is able to determine which of the plurality of targets are present and which are absent.
A method that identifies the particular base present at an interrogation position, optionally comprises a first probe, a second probe, a third probe, and a fourth probe. An interrogation position of the first probe comprises a nucleic acid residue that is a deoxyadenosine or adenosine residue. An interrogation position of the second probe comprises a nucleic acid residue that is a deoxythymidine or uridine residue. An interrogation position of the third probe comprises a nucleic acid residue that is a deoxyguanosine or guanosine residue. An interrogation position of the fourth nucleic acid probe comprises a nucleic acid residue that is a deoxycytosine or cytosine residue. Preferably, all four probes can be used in a single depolymerization reaction, and their released identifier nucleotides are distinguishable.
In another aspect of the invention, the sample containing a plurality of target nucleic acid sequences is admixed with a plurality of the nucleic acid probes. Several analytical outputs can be obtained from such multiplexed assays. In a first embodiment, the analytical output obtained when at least one nucleic acid probes hybridizes with partial complementarity to one target nucleic acid sequence is greater than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences. In a second embodiment, the analytical output obtained when at least one nucleic acid probe hybridizes with partial complementarity to one target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences. In a third embodiment, the analytical output obtained when at least one nucleic acid probe hybridizes with total complementarity to one nucleic acid target sequence is greater than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences. In a fourth embodiment, the analytical output obtained when at least one nucleic acid probe hybridizes with total complementarity to one target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences. The depolymerizing enzymes for use in these four embodiments are as described herein.
Yet another embodiment of the invention contemplates a method for determining the presence or absence of a first nucleic acid target in a nucleic acid sample that may contain that target or may contain a substantially identical second target. For example, the second target may have a single base substitution, deletion or addition relative to the first nucleic acid target. This embodiment comprises the following steps.
A sample to be assayed is admixed with a plurality of nucleic acid probes under hybridizing conditions to form a hybridization composition. The first and second nucleic acid targets each comprise a region of sequence identity except for at least a single nucleotide at a predetermined position that differs between the targets. Each of the nucleic acid probes is substantially complementary to a nucleic acid target region of sequence identity and comprises at least one identifier nucleotide at an interrogation position. An interrogation position of the probe is aligned with the predetermined position of a target when a target and probe are hybridized. Each probe also includes an identifier nucleotide in the 3xe2x80x2-terminal region.
The hybridization composition is maintained under hybridizing conditions for a time period sufficient to form a treated sample wherein the nucleotide at the interrogation position of the probe is aligned with the nucleotide at the predetermined position in the region of identity of the target.
A treated reaction mixture is formed by admixing the treated sample with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3xe2x80x2-terminus of a hybridized nucleic acid probe. The reaction mixture is maintained under depolymerization conditions for a time period sufficient to permit the enzyme to depolymerize the hybridized nucleic acid and release the identifier nucleotides.
An analytical output is obtained by analyzing for the presence or absence of identifier nucleotides released from the 3xe2x80x2 terminus of the hybridized probe. The analytical output indicates the presence or absence of released identifier nucleotide at the predetermined region, and; thereby, the presence or absence of a corresponding nucleic acid target.
One aspect of the above method is comprised of a first probe and a second probe in the same hybridization composition. The first probe comprises a nucleotide an interrogation position that is complementary to a first nucleic acid target at a predetermined position. The second probe comprises a nucleotide at an interrogation position that is complementary to a second nucleic acid target at a predetermined position.
Another aspect of the above method, the presence or absence of a third nucleic acid target, which is different from the first and second targets, is assayed for in the same sample that may further contain a fourth target that is substantially identical to the third target.
In one aspect of a process of the invention, the depolymerizing enzyme, whose activity is to release nucleotides, is a template-dependent polymerase, whose activity is to depolymerize hybridized nucleic acid, whose 3xe2x80x2-terminal nucleotide is matched, in the 3xe2x80x2 to 5xe2x80x2 direction in the presence of pyrophosphate ions to release one or more nucleotides. Thus, the enzyme""s activity is to depolymerize hybridized nucleic acid to release identifier nucleotides under depolymerizing conditions. Preferably, this enzyme depolymerizes hybridized nucleic acids whose bases in the 3xe2x80x2-terminal region of the probe are matched with total complementarity to the corresponding bases of the nucleic acid target. The enzyme will continue to release properly paired bases from the 3xe2x80x2-terminal region and will stop at or near the location where the enzyme arrives at a base that is mismatched.
In an alternative aspect of the process, the depolymerizing enzyme, whose activity is to release nucleotides, exhibits a 3xe2x80x2 to 5xe2x80x2 exonuclease activity in which hybridized nucleic acids having one or more mismatched bases at the 3xe2x80x2-terminus of the hybridized probe are depolymerized. Thus, the enzyme""s activity is to depolymerize hybridized nucleic acid to release nucleotides under depolymerizing conditions. In this embodiment, the hybrid may be separated from the free probe prior to enzyme treatment. In some embodiments, an excess of target may be used so that the concentration of free probe in the enzyme reaction is extremely low.
In still another alternative aspect of a process of the invention, the depolymerizing enzyme exhibits a 3xe2x80x2 to 5xe2x80x2 exonuclease activity on a double-stranded DNA substrate having one or more matched bases at the 3xe2x80x2 terminus of the hybrid. The enzyme""s activity is to depolymerize hybridized nucleic acid to release nucleotides containing a 5xe2x80x2 phosphate under depolymerizing conditions.
In a further aspect of the invention, the nucleic acid sample to be assayed is obtained from a biological sample that is a solid or liquid. Exemplary solid biological samples include animal tissues such as those obtained by biopsy or post mortem, and plant tissues such as leaves, roots, stems, fruit and seeds. Exemplary liquid samples include body fluids such as sputum, urine, blood, semen and saliva of an animal, or a fluid such as sap or other liquid obtained when plant tissues are cut or plant cells are lysed or crushed.
In one aspect of the method, the predetermined nucleic acid target sequence is a microbial or viral nucleic acid and nucleic acid probes comprise sequences complementary to those microbial or viral nucleic acid sequences.
In another aspect of the invention, the predetermined nucleic acid target sequence is a gene or region of a gene that is useful for genomic typing. Exemplary target sequences include the Leiden V mutation, a mutant xcex2-globin gene, the cystic fibrosis-related gene in the region of the delta 508 allele, a mutation in a prothrombin gene, congenital adrenal hyperplasia-associated genes, a translocation that takes place in the region of the bcr gene along with involvement of a segment of the abl gene, as well as the loss of heterozygosity of the locus of certain alleles as is found in certain cancers and also allelic trisomy.
Genomic typing can also be used to assay plant genomes such as that of rice, soy or maize, and the genomes of microbes such as Campylobacter jejuni, Listeria, E. coli OH157, and the genomes of viruses such as cytomegalovirus (CMV) or human immunodeficiency virus (HIV).
A still further embodiment of the invention contemplates a method using thermostable DNA polymerase as a depolymerizing enzyme for determining the presence or absence of a plurality of predetermined nucleic acid target sequences in a nucleic acid sample, and comprises the following steps.
A treated sample is provided that may contain a plurality of predetermined nucleic acid target sequences hybridized to their respective nucleic acid probes whose 3xe2x80x2-terminal regions are complementary to their predetermined nucleic acid target sequences and include an identifier nucleotide in the 3xe2x80x2-terminal region. A treated depolymerization reaction mixture is formed by admixing a treated sample with a depolymerizing amount of a enzyme whose activity is to release an identifier nucleotide from the 3xe2x80x2-terminus of a hybridized nucleic acid probe. In a preferred one-pot embodiment, the depolymerizing enzyme is thermostable and more preferably, the treated reaction mixture also contains (i) adenosine 5xe2x80x2 diphosphate, (ii) pyrophosphate, and (iii) a thermostable nucleoside diphosphate kinase (NDPK).
The treated sample is maintained under depolymerizing conditions at a temperature of about 4xc2x0 C. to about 90xc2x0 C., more preferably at a temperature of about 20xc2x0 C. to about 90xc2x0 C., and most preferably at a temperature of about 25xc2x0 C. to about 80xc2x0 C., for a time period sufficient to permit the depolymerizing enzyme to depolymerize the hybridized nucleic acid probe and release an identifier nucleotide as a nucleoside triphosphate. In preferred one-pot reactions, the time period is also sufficient to permit NDPK enzyme to transfer a phosphate from the released nucleoside triphosphate to added ADP, thereby forming ATP. The presence or absence of a nucleic acid target sequence is determined from the analytical output obtained using ATP. In a preferred method of the invention, analytical output is obtained by luminescence spectrometry.
In another aspect of the thermostable enzyme one-pot method for determining the presence or absence of a predetermined nucleic acid target sequence in a nucleic acid sample, the treated sample is formed by the following further steps. A hybridization composition is formed by admixing the sample to be assayed with a plurality of nucleic acid probes under hybridizing conditions. The 3xe2x80x2-terminal region of the nucleic acid probe (i) hybridizes with partial or total complementarity to a nucleic acid target sequence when that sequence is present in the sample, and (ii) includes an identifier nucleotide. A treated sample is formed by maintaining the hybridization composition under hybridizing conditions for a time period sufficient for the predetermined nucleic acid target sequence to hybridize with the nucleic acid probe.
Preferably, for the thermostable enzyme method, the depolymerizing enzyme is from a group of thermophilic DNA polymerases comprising Tne triple mutant DNA polymerase, Tne DNA polymerase, Taq DNA polymerase, Ath DNA polymerase, Tvu DNA polymerase, Bst DNA polymerase, and Tth DNA polymerase. In another aspect of the method, the NDPK is that encoded for by the thermophilic bacteria Pyrococcus furiosis (Pfu)
A still further embodiment of the invention contemplates determining the presence or absence of a plurality of nucleic acid target sequences in a nucleic acid sample using a plurality of special nucleic acid probes. These special probes hybridize to the target nucleic acid and are then modified to be able to form a hairpin structure. This embodiment comprises the following steps.
A treated sample is provided that contains a nucleic acid sample that may include a plurality of nucleic acid target sequences, each having an interrogation position, and each hybridized with its respective nucleic acid probe. The probes are comprised of at least two sections. The first section contains the probe 3xe2x80x2-terminal about 10 to about 30 nucleotides. These nucleotides are complementary to the target strand sequence at positions beginning about 1 to about 30 nucleotides downstream of the interrogation position. The second section of the probe is located at the 5xe2x80x2-terminal region of the probe and contains about 10 to about 20 nucleotides of the target sequence. This sequence spans the region in the target from the nucleotide at or just upstream (5xe2x80x2) of the interrogation position, to the nucleotide just upstream to where the 3xe2x80x2-terminal nucleotide of the probe anneals to the target. An optional third section of the probe, from zero to about 50, and preferably about zero to about 20 nucleotides in length and comprising a sequence that does not hybridize with either the first or second section, is located between the first and second sections of the probe.
The hybridized probes of the treated sample are extended in a template-dependent manner, as by admixture with dNTPs and a template-dependent polymerase, at least through the interrogation position, thereby forming an extended probe/target hybrid. In a preferred embodiment, the length of the probe extension is limited by omission from the extension reaction of a dNTP complementary to a nucleotide of the target sequence that is present upstream of the interrogation position and absent between the nucleotide complementary to the 3xe2x80x2-end of the interrogation position.
The extended probe/target hybrids are separated from any unreacted dNTPs. The extended probe/target hybrid is denatured to separate the strands. The extended probe strands are permitted to form hairpin structures.
A treated reaction mixture is formed by admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3xe2x80x2-terminus of an extended probe hairpin structure. The reaction mixture is maintained under depolymerizing conditions for a time period sufficient for the depolymerizing enzyme to release 3xe2x80x2-terminus nucleotides, and then analyzed for the presence of released identifier nucleotides. The analytical output indicates the presence or absence of the nucleic acid target sequences. In a preferred embodiment, the analytical output for the various targets are distinguishable.
A still further embodiment of the invention, termed REAPER(trademark), also utilizes hairpin structures. This method contemplates determining the presence or absence of a plurality of nucleic acid target sequences, or a specific base within a target sequence, in a nucleic acid sample, and comprises the following steps. A treated sample is provided that contains a nucleic acid sample that may include a plurality of nucleic acid target sequences hybridized with their respective first nucleic acid probe strands.
The hybrid is termed the first hybrid. The first probes are comprised of at least two sections. The first section contains the probe 3xe2x80x2-terminal about 10 to about 30 nucleotides that are complementary to the target nucleic acid sequence at a position beginning about 5 to about 30 nucleotides downstream of the target interrogation position. The second section of the first probe contains about 5 to about 30 nucleotides that are a repeat of the target sequence from the interrogation position to about 10 to about 30 nucleotides downstream of the interrogation position, and does not hybridize to the first section of the probe. An optional third section of the probe, located between the first and second sections of the probe, is zero to about 50, preferably up to about 20, nucleotides in length and comprises a sequence that does not hybridize to either the first or second section.
The first hybrid in the treated sample is extended at the 3xe2x80x2-end of the first probes, thereby extending the first probes past the interrogation position and forming an extended first hybrid whose sequence includes an interrogation position. The extended first hybrid is comprised of the original target nucleic acids and extended first probes. The extended first hybrid is then denatured in an aqueous composition to separate the two nucleic acid strands of the hybridized duplexes and form an aqueous solution containing separated target nucleic acids and a separated extended first probes.
Second probes that are about 10 to about 2000, preferably about 10 to about 200, most preferably about 10 to about 30 nucleotides in length and is complementary to the extended first probes at a position beginning about 5 to about 2000, preferably about 5 to about 200, nucleotides downstream of the interrogation position in extended first probe, is annealed to the extended first probe, thereby forming the second hybrid. The second hybrids is extended at the 3xe2x80x2-end of the second probes until that extension reaches the 5xe2x80x2-end of the extended first probes, thereby forming a second extended hybrid whose 3xe2x80x2-region includes an identifier nucleotide. In preferred embodiments the extending polymerase for both extensions does not add a nucleotide to the 3xe2x80x2 end that does not have a corresponding complementary nucleotide in the template.
An aqueous composition of the extended second hybrid is denatured to separate the two nucleic acid strands. The aqueous composition so formed is cooled to form a xe2x80x9chairpin structurexe2x80x9d from the separated extended second probes when its respective target sequence is present in the original nucleic acid sample.
A treated reaction mixture is formed by admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3xe2x80x2-terminus of a nucleic acid hybrid. The reaction mixture is maintained under depolymerizing conditions for a time period sufficient to release 3xe2x80x2-terminal region identifier nucleotides, and then analyzed for the presence of released identifier nucleotide. The analytical output indicates the presence or absence of the various nucleic acid target sequences.
The present invention has many benefits and advantages, several of which are listed below.
One benefit of the invention is that, in some embodiments, a plurality of nucleic acid hybrids can be detected with very high levels of sensitivity without the need for radiochemicals or electrophoresis.
An advantage of the invention is that the presence or absence of a plurality of target nucleic acid(s) can be detected reliably, reproducibly, and with great sensitivity.
A further benefit of the invention is that quantitative information can be obtained about the amount of a target nucleic acid sequence present in a sample.
A further advantage of the invention is that very slight differences in nucleic acid sequence are detectable, including single nucleotide polymorphisms (SNPs).
Yet another benefit of the invention is that the presence or absence of a number of target nucleic acid sequences can be determined in the same assay.
Yet another advantage of the invention is that the presence or absence of target nucleic acids can be determined with a small number of reagents and manipulations.
Another benefit of the invention is that the processes lend themselves to automation.
Still another benefit of the invention is its flexibility of use in many different types of applications and assays including, but not limited to, detection of mutations, translocations, and SNPs in nucleic acid (including those associated with genetic disease), determination of viral load, species identification, sample contamination, and analysis of forensic samples.
Still further benefits and advantages of the invention will become apparent from the specification and claims that follow.